Prostaglandin e1 and e2 analogs for the treatment of various medical conditions

ABSTRACT

A prostaglandin analog with selectivity to EP receptors and demonstrating EP agonist activity that may be used to expand hematopoietic stem cell populations or to treat or prevent influenza, bone fracture, bone disease, glaucoma, ocular hypertension, dysmenorrhoea, pre-term labor, immune disorders, osteoporosis, asthma, allergy, male sexual dysfunction, female sexual dysfunction, periodontal disease, gastric ulcer, renal disease, or other EP receptor-mediated conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention claims priority from U.S. Provisional ApplicationNo. 60/987,859 filed Nov. 14, 2007 entitled “Prostaglandin E₁ and E₂Derivatives as Selective E₂ Agonists for Medicinal Treatment,” and U.S.Provisional Application No. 61/037,493 filed Mar. 18, 2008 entitled“Prostaglandin E₁ and E₂ Derivatives as Selective E₂ Agonists forMedicinal Treatment.”

FIELD OF THE INVENTION

The present invention relates to pharmaceutically active compounds andmore particularly to prostaglandin analogs with selectivity forprostaglandin E (EP) receptors and demonstrating EP agonist activity,and the use of such compounds and compositions thereof for the treatmentof various medical conditions.

BACKGROUND OF THE INVENTION

Prostanoids are ubiquitous lipid mediator biomolecules involved innumerous physiological processes, such as the contraction and relaxationof smooth muscle, vasodilation, vasoconstriction, pain, regulation ofblood pressure, and modulation of inflammation. Prostanoids are a familyof eicosanoids that comprise prostaglandins (PGs), prostacyclins (PGIs),and thromboxanes (Txs). Their receptors belong to the G-protein coupledreceptor (GPCR) superfamily of receptors and may be grouped into fiveclasses, namely, prostaglandin D (DP), prostaglandin E (EP),prostaglandin F (FP), prostaglandin I (IP), and Thromboxane A (TP) basedon their sensitivity to five naturally occurring prostanoids, PGD₂,PGE₂, PGF_(2α), PGI₂, and TxA₂, respectively (Coleman, R. A., ProstanoidReceptors. IUPHAR compendium of receptor characterization andclassification, 2^(nd) edition, 338-353, 2000). EP receptors have beencharacterized into four subtypes EP₁, EP₂, EP₃, and EP₄. Each subtypehas been cloned and is distinct at both a molecular and pharmacologicallevel.

Prostanoids are synthesized from essential fatty acids comprising twentycarbon atoms, such as arachidonic acid and 8,11,14-eicosatrienoic acid.Prostanoids are synthesized in response to both extracellular andintracellular stimuli and are then rapidly released from the cells. Ingeneral, the short half-lives of most prostanoids ensure they act nearthe sites of their biosynthesis.

Prostaglandin E₂ (PGE₂) is a potent endogenous EP receptor agonistderived from arachidonic acid, shown below, and possesses twocarbon-carbon double bonds, one in each the α-chain and co-chain, and isthus called a “Series 2” prostaglandin.

Prostaglandin E₁ (PGE₁) is derived from 8,11,14-eicosatrienoic acid andpossesses only one carbon-carbon double bond, located in the co-chain,and is thus called a “Series 1” prostaglandin.

Both prostanoid and non-prostanoid EP receptor agonists are known. EPreceptor agonists may have a number of utilities. These include, but arenot limited to treatment of influenza (WO 2008/058766), bone fracturehealing (Li, M., et al., J. Bone Miner. Res., 18(11), 2003, 2033-2042;Paralkar, V. M., PNAS, 100(11), 2003, 6736-6740; WO 2002/24647; WO1998/27976), bone disease (WO 2002/24647), glaucoma (WO 2008/015517; WO2007/027468; WO 2003/040126), ocular hypertension (WO 2003/040126),dysmenorrhoea (WO 2003/037433), pre-term labor (GB 2 293 101), immunedisorders (WO 2003/037433), osteoporosis (WO 1998/27976; WO 2001/46140),asthma (WO 2003/037433), allergy (WO 2003/037433), fertility (Breyer, R.M., et al., Ann. N.Y. Acad. Sci., 905, 2000, 221-231), male sexualdysfunction (WO 2000/40248), female sexual dysfunction (U.S. Pat. No.6,562,868), periodontal disease (WO 2000/31084), gastric ulcer (U.S.Pat. No. 5,576,347), and renal disease (WO 1998/34916). EP receptoragonists may also be useful for expansion of hematopoietic stem cellpopulations (WO 2008/073748; North, T. E., et al., Nature, 447, 200 7,1007-1011).

SUMMARY OF THE INVENTION

The exemplary embodiments may be directed to compounds of structuralformula (I) that may be used to expand hematopoietic stem cellpopulations or to treat or prevent influenza, bone fracture, bonedisease, glaucoma, ocular hypertension, dysmenorrhoea, pre-term labor,immune disorders, osteoporosis, asthma, allergy, male sexualdysfunction, female sexual dysfunction, periodontal disease, gastriculcer, renal disease, or other EP receptor-mediated conditions whereinC⁹, C¹¹, R¹, Z¹, Z², Z³, Z⁴, Z¹, Z⁶, and Z⁷ are defined herein:

Another aspect of the embodiment is a pharmaceutical compositioncomprising a pharmaceutically effective amount of a compound accordingto formula (I), any stereoisomer or geometric isomer thereof, or aprodrug thereof, or a hydrate or solvate thereof, or a pharmaceuticallyacceptable salt thereof, in admixture with a pharmaceutically acceptablecarrier.

Another aspect of the embodiment is directed to a method of expandinghematopoietic stem cell populations in a culture or patient in needthereof by administering to the culture or patient a compound accordingto formula (I), any stereoisomer or geometric isomer thereof, or aprodrug thereof, or a hydrate or solvate thereof, or a pharmaceuticallyacceptable salt thereof.

Another aspect of the embodiment is directed to a method of treating orpreventing influenza, bone fracture, bone disease, glaucoma, ocularhypertension, dysmenorrhoea, pre-term labor, immune disorders,osteoporosis, asthma, allergy, male sexual dysfunction, female sexualdysfunction, periodontal disease, gastric ulcer, renal disease, or otherEP receptor-mediated conditions in a patient in need thereof byadministering to the patient a compound according to formula (I), anystereoisomer or geometric isomer thereof, or a prodrug thereof, or ahydrate or solvate thereof, or a pharmaceutically acceptable saltthereof.

Other exemplary embodiments of the invention will become apparent fromthe detailed description provided hereinafter. It should be understoodthat the detailed description and specific examples, while disclosingexemplary embodiments of the invention, are intended for purposes ofillustration only and are not intended to limit the scope of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

The exemplary embodiments are directed to a compound of formula (I),their preparation, pharmaceutical compositions comprising thesecompounds, and their pharmaceutical use in the prevention and treatmentof EP receptor-mediated diseases or conditions. The compounds of formula(I) are shown below:

wherein:dashed bonds may each independently represent a second carbon-carbonbond in order to give a carbon-carbon double bond with either (E) or (Z)geometry or may be ignored in order to give a carbon-carbon single bond;C⁹ and C¹¹ each is independently C═CH₂, C═O, CF₂, CHF (anystereoisomer), or C(H)OH (any stereoisomer) with the proviso that C⁹does not equal C¹¹, and also with the proviso that when one of either C⁹or C¹¹ is C═O, and the other is C(H)OH, at least one of Z², Z³, Z⁴, andZ⁵ is fluorine, and also with the proviso that when one of either C⁹ orC¹¹ is CHF, the other is not C(H)OH;R¹ is CO₂R³, CH₂OR³, CONR⁴R⁵, COCH₂OH, CONR⁴SO₂R⁵, P(O)(OR⁴)₂, or

R³ is hydrogen or (C₁-C₆)-alkyl;R⁴ and R⁵ each is independently hydrogen or (C₁-C₆)-alkyl;Z¹ are hydrogen or fluorine;Z² and Z³ each is independently hydrogen or fluorine;Z⁴ and Z⁵ each is independently hydrogen, fluorine, hydroxy, or methyl,or together are an oxygen atom that form a carbonyl group with theadjoining carbon atom of the ω chain;Z⁶ and Z⁷ each is independently hydrogen, fluorine, hydroxy, or methyl,or together are an oxygen atom that form a carbonyl group with theadjoining carbon atom of the ω chain;

The exemplary embodiment above may also include any stereoisomer orgeometric isomer thereof, or an equivalent thereof, or a prodrugthereof, or a hydrate or solvate thereof, or a pharmaceuticallyacceptable salt thereof.

Another exemplary embodiment may be directed to a compound of formula(I) wherein C⁹ is C═O and C¹¹ is C═CH₂.

Another exemplary embodiment may be directed to a compound of formula(I) wherein C⁹ is C(H)OH and C¹¹ is C═CH₂.

Another exemplary embodiment may be directed to a compound of formula(I) wherein C⁹ is C═CH₂ and C¹¹ is C═O.

Another exemplary embodiment may be directed to a compound of formula(I) wherein C⁹ is C═O and C¹¹ is CF₂.

Another exemplary embodiment may be directed to a compound of formula(I) wherein C⁹ is C(H)OH and C¹¹ is CF₂.

Another exemplary embodiment may be directed to a compound of formula(I) wherein C⁹ is CF₂ and C¹¹ is C═O.

Another exemplary embodiment may be directed to a compound of formula(I) wherein C⁹ is CF₂ and C¹¹ is C(H)OH.

Another exemplary embodiment may be directed to a compound of formula(I) wherein C⁹ is C═O and C¹¹ is CHF.

Another exemplary embodiment may be directed to a compound of formula(I) wherein C⁹ is CHF and C¹¹ is C═O.

Another exemplary embodiment may be directed to a compound of formula(I) wherein R¹ is CO₂H.

Another exemplary embodiment may be directed to a compound of formula(I) wherein R¹ is CO₂ ^(i)Pr.

Another exemplary embodiment may be directed to a compound of formula(I) wherein R¹ is CON(H)Et.

Another exemplary embodiment may be directed to a compound of formula(I) wherein R¹ is CON(H)SO₂Me.

Another exemplary embodiment may be directed to a compound of formula(I) wherein R¹ is CH₂OH.

Another exemplary embodiment may be directed to a compound of formula(I) wherein R¹ is

Another exemplary embodiment may be directed to a compound of formula(I) wherein Z¹ is hydrogen.

Another exemplary embodiment may be directed to a compound of formula(I) wherein Z¹ is fluorine.

Another exemplary embodiment may be directed to a compound of formula(I) wherein Z² is fluorine and Z³ is hydrogen.

Another exemplary embodiment may be directed to a compound of formula(I) wherein Z² is hydrogen and Z³ is fluorine.

Another exemplary embodiment may be directed to a compound of formula(I) wherein each Z⁴ and Z⁵ is fluorine.

Another exemplary embodiment may be directed to a compound of formula(I) wherein each Z⁴ and Z⁵ is methyl.

Another exemplary embodiment may be directed to a compound of formula(I) wherein Z⁴ is hydroxy and Z⁵ is methyl.

Another exemplary embodiment may be directed to a compound of formula(I) wherein Z⁴ and Z⁵ together is an oxygen atom that form a carbonylwith the adjoining carbon atom.

Another exemplary embodiment may be directed to a compound of formula(I) wherein each Z⁶ and Z⁷ is hydrogen.

Another exemplary embodiment may be directed to a compound of formula(I) wherein each Z⁶ and Z⁷ is fluorine.

Another exemplary embodiment may be directed to a compound of formula(I) wherein each Z⁶ and Z⁷ is methyl.

Another exemplary embodiment may be directed to a compound of formula(I) wherein Z⁶ is hydroxy and Z⁷ is hydrogen.

Another exemplary embodiment may be directed to a compound of formula(I) wherein Z⁶ is hydroxy and Z⁷ is methyl.

Another exemplary embodiment may be directed to a compound of formula(I) wherein Z⁶ is methyl and Z⁷ is hydrogen.

Another exemplary embodiment may be directed to a compound of formula(I) wherein Z⁶ and Z⁷ together is an oxygen atom that form a carbonylwith the adjoining carbon atom.

Another exemplary embodiment may be directed to a more specificembodiment of the compound of formula (I), namely to a compound offormula (I):

or an equivalent thereof, or a hydrate or solvate thereof, or apharmaceutically acceptable salt thereof.

Another exemplary embodiment may be directed to a more specificembodiment of the compound of formula (I), namely to a compound offormula (III):

or an equivalent thereof, or a hydrate or solvate thereof, or apharmaceutically acceptable salt thereof.

Another exemplary embodiment may be directed to a more specificembodiment of the compound of formula (I), namely to a compound offormula (IV):

or an equivalent thereof, or a hydrate or solvate thereof, or apharmaceutically acceptable salt thereof.

Another exemplary embodiment may be directed to a more specificembodiment of the compound of formula (I), namely to a compound offormula (V):

or an equivalent thereof, or a hydrate or solvate thereof, or apharmaceutically acceptable salt thereof.

Another exemplary embodiment may be a compound selected from the groupconsisting of:(Z)-2,2-difluoro-7-((1R,2R)-2-((S,E)-3-hydroxyoct-1-enyl)-3-methylene-5-oxocyclopentyl)hept-5-enoicacid;(Z)-7-((1R,2R)-2-((1R,3S)-1-fluoro-3-hydroxyoctyl)-3-methylene-5-oxocyclopentyl)hept-5-enoicacid;(Z)-7-((1R,2R)-2-((1S,3S)-2-fluoro-3-hydroxyoctyl)-3-methylene-5-oxocyclopentyl)hept-5-enoicacid;(Z)-7-((1R,2R)-2-((2R,3S)-2-fluoro-3-hydroxyoctyl)-3-methylene-5-oxocyclopentyl)hept-5-enoicacid;(Z)-7-((1R,2R)-2-((2S,3S)-2-fluoro-3-hydroxyoctyl)-3-methylene-5-oxocyclopentyl)hept-5-enoicacid;(Z)-7-((1R,2R)-2-((E)-3,3-difluorooct-1-enyl)-3-methylene-5-oxocyclopentyl)hept-5-enoicacid;(Z)-7-((1R,2R)-2-((R,E)-3-hydroxy-3-methyloct-1-enyl)-3-methylene-5-oxocyclopentyl)hept-5-enoicacid;(Z)-7-((1R,2R)-2-((R,E)-3-hydroxy-4,4-dimethyloct-1-enyl)-3-methylene-5-oxocyclopentyl)hept-5-enoicacid;(Z)-7-((1R,2R)-2-((R,E)-4,4-difluoro-3-hydroxyoct-1-enyl)-3-methylene-5-oxocyclopentyl)hept-5-enoicacid; and(Z)-7-((1R,2R)-2-((S,E)-3-hydroxy-3-methyloct-1-enyl)-3-methylene-5-oxocyclopentyl)hept-5-enoicacid; or an equivalent thereof, or a (C₁-C₆)-alkyl ester thereof, or anN—(C₁-C₆)-alkyl amide thereof, or an N-methylsulfonyl amide thereof, ora hydrate, solvate, or a pharmaceutically acceptable salt thereof.

Another exemplary embodiment may be a compound selected from the groupconsisting of:(Z)-7-((1R,2R)-2-((E)-3,3-difluorooct-1-enyl)-5-methylene-3-oxocyclopentyl)hept-5-enoicacid;(Z)-7-((1R,2R)-3,3-difluoro-2-((S,E)-3-hydroxy-3-methyloct-1-enyl)-5-oxocyclopentyl)hept-5-enoicacid;(Z)-7-((1R,2R,3R)-2-((E)-3,3-difluorooct-1-enyl)-3-hydroxy-5-methylenecyclopentyl)hept-5-enoicacid;(Z)-7-((1R,2R,3R)-3-fluoro-2-((R,E)-3-hydroxy-4,4-dimethyloct-1-enyl)-5-oxocyclopentyl)hept-5-enoicacid;(Z)-7-((1R,2R,3S)-3-fluoro-2-((R,E)-3-hydroxy-4,4-dimethyloct-1-enyl)-5-oxocyclopentyl)hept-5-enoicacid;(Z)-7-((1R,2R,5R)-5-fluoro-2-((R,E)-3-hydroxy-4,4-dimethyloct-1-enyl)-3-oxocyclopentyl)hept-5-enoicacid;(Z)-7-((1R,2R,5S)-2-((E)-3,3-difluorooct-1-enyl)-5-hydroxy-3-methylenecyclopentyl)hept-5-enoicacid;(Z)-7-((1R,2R,5S)-3,3-difluoro-5-hydroxy-2-((R,E)-3-hydroxy-4,4-dimethyloct-1-enyl)cyclopentyl)hept-5-enoicacid;(Z)-7-((1R,2R,5S)-5-fluoro-2-((R,E)-3-hydroxy-4,4-dimethyloct-1-enyl)-3-oxocyclopentyl)hept-5-enoicacid;(Z)-7-((1R,4R,5R)-2,2-difluoro-4-hydroxy-5-((R,E)-3-hydroxy-4,4-dimethyloct-1-enyl)cyclopentyl)hept-5-enoicacid; and(Z)-7-((1R,5R)-2,2-difluoro-5-((S,E)-3-hydroxy-3-methyloct-1-enyl)-4-oxocyclopentyl)hept-5-enoicacid; or an equivalent thereof, or a (C₁-C₆)-alkyl ester thereof, or anN—(C₁-C₆)-alkyl amide thereof, or an N-methylsulfonyl amide thereof, ora hydrate, solvate, or a pharmaceutically acceptable salt thereof.

The exemplary embodiments may also be directed to a method of preventingor treating a disease or condition mediated at least in part by agonismof an EP receptor, in a subject in need of such treatment, comprisingadministering to the subject a therapeutically effective amount of acompound of any exemplary embodiment of formula (I), or apharmaceutically acceptable salt, hydrate, or solvate thereof; the useof a compound of any exemplary embodiment of formula (I), or apharmaceutically acceptable salt, hydrate, or solvate thereof, for themanufacture of a medicament for preventing or treating a disease orcondition mediated at least in part by agonism of an EP receptor; acompound of any exemplary embodiment of formula (I), or apharmaceutically acceptable salt, hydrate, or solvate thereof, for useas a medicament; a compound of any exemplary embodiment of formula (I),or a pharmaceutically acceptable salt, hydrate, or solvate thereof, foruse in the prevention or treatment of a disease or condition mediated atleast in part by agonism of an EP receptor; a pharmaceutical compositioncomprising a compound of any exemplary embodiment of formula (I), or apharmaceutically acceptable salt, hydrate, or solvate thereof, and apharmaceutically acceptable excipient; a pharmaceutical composition forthe prevention and treatment of a disease or condition mediated at leastin part by agonism of an EP receptor comprising a compound of anyexemplary embodiment of formula (I), or a pharmaceutically acceptablesalt, hydrate, or solvate thereof.

The diseases and conditions mediated at least in part by agonism of anEP receptor may include allergy and allergic inflammation. Diseases andconditions of this kind may be allergic respiratory conditions such asallergic rhinitis, nasal congestion, rhinorrhea, perennial rhinitis,nasal inflammation, asthma of all types, chronic obstructive pulmonarydisease (COPD), chronic or acute bronchoconstriction, chronicbronchitis, small airways obstruction, emphysema, chronic eosinophilicpneumonia, adult respiratory distress syndrome, exacerbation of airwayshyper-reactivity consequent to other drug therapy, airways disease thatmay be associated with pulmonary hypertension, acute lung injury,bronchiectasis, sinusitis, allergic conjunctivitis, or atopicdermatitis, particularly asthma or chronic obstructive pulmonarydisease.

Types of asthma may include atopic asthma, non-atopic asthma, allergicasthma, atopic bronchial IgE-mediated asthma, bronchial asthma,essential asthma, true asthma, intrinsic asthma caused bypathophysiologic disturbances, extrinsic asthma caused by environmentalfactors, essential asthma of unknown or inapparent cause, bronchiticasthma, emphysematous asthma, exercise-induced asthma, exertion asthma,allergen-induced asthma, cold air induced asthma, occupational asthma,infective asthma caused by bacterial, fungal, protozoal, or viralinfection, non-allergic asthma, incipient asthma, wheezy infantsyndrome, and bronchiolytis.

Included in the use of the compounds of any exemplary embodiment offormula (I) for the treatment of asthma, may be palliative treatment forthe symptoms and conditions of asthma such as wheezing, coughing,shortness of breath, tightness in the chest, shallow or fast breathing,nasal flaring (nostril size increases with breathing), retractions (neckarea and between or below the ribs moves inward with breathing),cyanosis (gray or bluish tint to skin, beginning around the mouth),runny or stuffy nose, and headache.

The exemplary embodiments may also be directed to any of the uses,methods, or compositions as defined above wherein the compound of anyexemplary embodiment of formula (I), or a pharmaceutically acceptablesalt, hydrate, or solvate thereof, may be used in combination withanother pharmacologically active compound. Specific combinations usefulfor the treatment of allergy or asthma may include combinationscomprising a compound of formula (I), or a pharmaceutically acceptablesalt, hydrate, or solvate thereof, and (i) a glucocorticosteroid or DAGR(dissociated agonist of the corticoid receptor); (ii) a β₂ agonist, anexample of which is a long-acting β₂ agonist; (iii) a muscarinic M3receptor antagonist or anticholinergic agent; (iv) a histamine receptorantagonist or inverse agonist, which may be an H1 or an H3 antagonist orinverse agonist; (v) a 5-lipoxygenase inhibitor; (vi) a thromboxaneinhibitor; (vii) an LTD₄ inhibitor; (viii) a kinase inhibitor; or (ix) avaccine. Generally, the compounds of the combination may be administeredtogether as a formulation in association with one or morepharmaceutically acceptable excipients.

Other diseases and conditions that may be mediated, at least in part, byagonism of an EP receptor are influenza, bone fracture healing, bonedisease, glaucoma, ocular hypertension, dysmenorrhoea, pre-term labor,immune disorders, osteoporosis, asthma, allergy, fertility, male sexualdysfunction, female sexual dysfunction, periodontal disease, gastriculcer, and renal disease. EP receptor agonists may also be useful forexpansion of hematopoietic stem cell populations.

Besides being useful for human treatment, compounds of formula (I) mayalso be useful for veterinary treatment of companion animals, exoticanimals, and farm animals.

When used in the present application, the following abbreviations havethe meaning set out below: Ac is acetyl; ACN is acetonitrile; BBr₃ isboron tribromide; Bn is benzyl; BnNH₂ is benzylamine; BSA is bovineserum albumin; CH₂Cl₂ is dichloromethane; CHCl₃ is chloroform; CDCl₃ isdeuterochloroform; DAST is diethylaminosulfur trifluoride; DCC isN,N′-dicyclohexylcarbodiimide; DCM is dichloromethane; DIBAL-Hisdiisobutylaluminum hydride; DME is 1,2-dimethoxyethane; DMEM isDulbecco's minimal essential medium; DMF is N,N-dimethylformamide; DMSOis dimethyl sulfoxide; DBU is 1,8-diazabicyclo[5.4.0]undec-7-ene;EDC/EDAC is N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride; EDTA is ethylenediaminetetraacetic acid; EIA is enzymeimmunoassay; Et is ethyl; Et₃N is triethylamine; HOBt is1-hydroxybenzotriazole; HBSS is Hank's balanced salt solution; IBMX isisobutylmethylxanthine; ^(i)Pr is isopropyl; MCS is multiple cloningsite; Me is methyl; MES is 2-(N-morpholino)ethanesulfonic acid; NaHMDSis sodium hexamethyldisilazane, also known as sodiumbis(trimethylsilyl)amide; NMP is 1-methyl-2-pyrrolidinone; PCR ispolymerase chain reaction; Ph is phenyl; Pd(PPh₃)₄ istetrakis(triphenylphosphine)palladium; PhB(OH)₂ is benzeneboronic acid,also known as phenylboronic acid; PhMe is toluene; rt is roomtemperature; TBAF is tetrabutylammonium fluoride; t-Bu is tert-butyl;TCA is trichloroacetic acid; THF is tetrahydrofuran; and Tris-HCl is2-amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride.

Unless otherwise defined herein, scientific and technical terms used inconnection with the exemplary embodiments shall have the meanings thatare commonly understood by those of ordinary skill in the art.

Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.Generally, nomenclature used in connection with, and techniques ofchemistry and molecular biology described herein are those well knownand commonly used in the art.

The phrase “therapeutically effective” is intended to qualify the amountof compound or pharmaceutical composition, or the combined amount ofactive ingredients in the case of combination therapy. This amount orcombined amount may achieve the goal of treating the relevant condition.

The term “treatment,” as used herein to describe the exemplaryembodiments and unless otherwise qualified, means administration of thecompound, pharmaceutical composition, or combination to effectpreventative, palliative, supportive, restorative, or curativetreatment. The term treatment encompasses any objective or subjectiveimprovement in a subject with respect to a relevant condition ordisease.

The term “preventative treatment,” as used herein to describe theexemplary embodiments, means that the compound, pharmaceuticalcomposition, or combination may be administered to a subject to inhibitor stop the relevant condition from occurring in a subject, particularlyin a subject or member of a population that may be significantlypredisposed to the relevant condition.

The term “palliative treatment,” as used herein to describe theexemplary embodiments, means that the compound, pharmaceuticalcomposition, or combination may be administered to a subject to remedysigns and/or symptoms of a condition, without necessarily modifying theprogression of, or underlying etiology of, the relevant condition.

The term “supportive treatment,” as used herein to describe theexemplary embodiments, means that the compound, pharmaceuticalcomposition, or combination may be administered to a subject as part ofa regimen of therapy, but that such therapy is not limited toadministration of the compound, pharmaceutical composition, orcombination. Unless otherwise expressly stated, supportive treatment mayembrace preventative, palliative, restorative, or curative treatment,particularly when the compounds or pharmaceutical compositions arecombined with another component of supportive therapy.

The term “restorative treatment,” as used herein to describe theexemplary embodiments, means that the compound, pharmaceuticalcomposition, or combination may be administered to a subject to modifythe underlying progression or etiology of a condition. Non-limitingexamples include an increase in forced expiratory volume in one second(FEV 1) for lung disorders, inhibition of progressive nerve destruction,reduction of biomarkers associated and correlated with diseases ordisorders, a reduction in relapses, improvement in quality of life, andthe like.

The term “curative treatment,” as used herein to describe the exemplaryembodiments, means that the compound, pharmaceutical composition, orcombination may be administered to a subject for the purpose of bringingthe disease or disorder into complete remission, or that the disease ordisorder in undetectable after such treatment.

The term “alkyl,” alone or in combination, means an acyclic radical,linear or branched, preferably containing from 1 to about 6 carbonatoms. Examples of such radicals include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl,hexyl, heptyl, octyl, and the like. Where no specific substitution isspecified, alkyl radicals may be optionally substituted with groupsconsisting of hydroxy, sulfhydryl, methoxy, ethoxy, amino, cyano,chloro, and fluoro.

The carbon atom content of various hydrocarbon-containing moieties isindicated by a prefix designating a lower and upper number of carbonatoms in the moiety, that is, the prefix C_(i)-C_(j) indicates a moietyof the integer “i” to the integer “j” carbon atoms, inclusive. Thus, forexample, ‘(C₁-C₈)-alkyl’ refers to alkyl of one to eight carbon atoms,inclusive.

The terms “hydroxy” and “hydroxyl,” as used herein, mean an OH radical.

The term “sulfhydryl,” as used herein, means an SH radical.

The term “oxo” means a doubly bonded oxygen.

The term “alkoxy” means a radical comprising an alkyl radical that isbonded to an oxygen atom, such as a methoxy radical.

The term “aryl” means a fully unsaturated mono- or multi-ring cycloalkylhaving a cyclic array of p-orbitals containing 4n+2 electrons,including, but not limited to, substituted or unsubstituted phenyl,naphthyl, or anthracenyl optionally fused to a carbocyclic radicalwherein aryl may be optionally substituted with one or more substituentsfrom the group consisting of halo, methoxy, ethoxy, (C₁-C₆)-alkyl,phenyl, O-phenyl, cyano, nitro, hydroxyl, sulfhydryl, ortrifluoromethyl.

The term “halo,” as used herein, means one of the following groupconsisting of fluoro, chloro, bromo, or iodo.

The terms “heterocycle”, “heterocyclic ring system,” and “heterocyclyl”refer to a saturated or unsaturated mono- or multi-ring cycloalkylwherein one or more carbon atoms is replaced by N, S, or O. The terms“heterocycle”, “heterocyclic ring system,” and “heterocyclyl” includefully saturated ring structures such as piperazinyl, dioxanyl,tetrahydrofuranyl, oxiranyl, aziridinyl, morpholinyl, pyrrolidinyl,piperidinyl, thiazolidinyl, and others. The terms “heterocycle”,“heterocyclic ring system,” and “heterocyclyl” also include partiallyunsaturated ring structures such as dihydrofuranyl, pyrazolinyl,imidazolinyl, pyrrolinyl, chromanyl, dihydrothiphenyl, and others.

The term “heteroaryl” refers to an aromatic heterocyclic group.Heteroaryl is preferably: (a) a five-membered aromatic heterocyclicgroup containing either (i) 1-4 nitrogen atoms or (ii) 0-3 nitrogenatoms and 1 oxygen or 1 sulfur atom; (b) a six-membered aromaticheterocyclic group containing 1-3 nitrogen atoms; (c) a nine-memberedbicyclic heterocyclic group containing either (i) 1-5 nitrogen atoms or(ii) 0-4 nitrogen atoms and 1 oxygen or 1 sulfur atom; or (d) aten-membered bicyclic aromatic heterocyclic group containing 1-6nitrogen atoms; each of said groups (a)-(d) being optionally substitutedby one or more of (C₁-C₆)-alkyl, (C₁-C₆)-fluoroalkyl,(C₃-C₆)-cycloalkyl, hydroxy(C₃-C₆)-cycloalkyl, (C₂-C₆)-alkenyl,(C₂-C₆)-alkynyl, halo, oxo, hydroxyl, (C₁-C₆)-alkoxy, sulfhydryl, —SMe,or cyano. Examples of “heteroaryl” include pyridyl, pyrimidinyl,pyridazinyl, pyrazinyl, thionyl, furanyl, pyrrolyl, pyrazolyl,imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl,oxadiazolyl, thiadiazolyl, and tetrazolyl, optionally substituted asspecified above.

In “heterocycle” or “heteroaryl,” the point of attachment to themolecule of interest may be at a heteroatom or elsewhere within thering.

The term “cycloalkyl” means a mono- or multi-ringed cycloalkyl whereineach ring contains three to ten carbon atoms, preferably three to sixcarbon atoms. “Cycloalkyl” is preferably a monocyclic cycloalkylcontaining from three to six carbon atoms. Examples include cyclopropyl,cyclobutyl, cyclopentyl, and cyclohexyl.

The term “excipient” is used herein to describe any ingredient otherthan a compound of formula (I). The choice of excipient will to a largeextent depend on factors such as the particular mode of administration,the effect of the excipient on solubility and stability, and the natureof the dosage form. The term “excipient” encompasses diluents, carrier,or adjuvant.

Pharmaceutically acceptable salts of the compounds of formula (I)include the acid addition and base salts thereof.

Suitable acid addition salts are formed by acids which form non-toxicsalts. Examples include the acetate, adipate, aspartate, benzoate,besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate,citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate,gluconate, glucuronate, hexafluorophosphate, hibenzate,hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide,isethionate, lactate, malate, maleate, malonate, mesylate,methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate,oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogenphosphate, propionate, pyroglutamate, saccharate, stearate, succinate,tannate, tartrate, tosylate, trifluoroacetate,naphthalene-1,5-disulfonic acid, and xinofoate salts.

Suitable base salts are formed from bases which form non-toxic salts.Examples include the aluminum, arginine, benzathine, calcium, choline,diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine,potassium, sodium, tromethamine, and zinc salts.

Hemisalts of acids and bases may also be formed, for example,hemisulfate and hemicalcium salts. For a review on suitable salts, seeHandbook of Pharmaceutical Salts: Properties, Selection, and Use, byStahl and Wermuth (Wiley-VCH, 2002).

The compounds of any exemplary embodiment of formula (I) may also existin unsolvated and solvated forms. The term “solvate” is used herein todescribe a molecular complex comprising the compound of any exemplaryembodiment of formula (I), or a pharmaceutically acceptable saltthereof, and one or more pharmaceutically acceptable solvent molecules,for example, ethanol. The term “hydrate” is employed when said solventis water.

Also included herein are multi-component complexes other than salts andsolvates wherein the compound of formula (I) and at least one othercomponent are present in stoichiometric or non-stoichiometric amounts.

The compounds of any exemplary embodiment of formula (I) may exist in acontinuum of solid states ranging from fully amorphous to fullycrystalline.

The compounds of any exemplary embodiment of formula (I) may also existin a mesomorphic state (mesophase or liquid crystal) when subjected tosuitable conditions. The mesomorphic state is intermediate between thetrue crystalline state and the true liquid state (either melt orsolution).

Hereinafter all references to compounds of any exemplary embodiment offormula (I) include references to salts, solvates, multi-componentcomplexes, and liquid crystals thereof and to solvates, multi-componentcomplexes, and liquid crystals of salts thereof.

Also included herein are all polymorphs and crystal habits of compoundsof any exemplary embodiment of formula (I), prodrugs, and isomersthereof (including optical, geometric, and tautomeric isomers) andisotopically-labeled forms thereof.

Compounds of any exemplary embodiment of formula (I) may be administeredorally, topically, transdermally, intranasally, by inhalation, directlyinto the bloodstream, into muscle, into an internal organ, into the eye,into the ear, into the rectum, or by other means.

The compounds herein, their methods or preparation and their biologicalactivity will appear more clearly from the examination of the followingexamples that are presented as an illustration only and are not to beconsidered as limiting the invention in its scope. Compounds herein areidentified, for example, by the following analytical methods.

Mass spectra (MS) methods include positive electrospray ionization(ESI⁺), negative electrospray ionization (ESI⁻), positive atmosphericpressure chemical ionization (APCI⁺), or negative atmospheric pressurechemical ionization (APCI⁻).

300 MHz proton nuclear magnetic resonance spectra (¹H NMR) are recordedat ambient temperature using a Bruker (300 MHz) spectrometer. In the ¹HNMR chemical shifts (δ) are indicated in parts per million (ppm) withreference to tetramethylsilane (TMS) as the internal standard.

The above description of embodiments of the invention is merelyexemplary in nature and, thus, variations thereof are not to be regardedas a departure from the spirit and scope of the invention.

EXAMPLES Example 1 Preparation of(Z)-7-((1R,2R)-2-((E)-3-hydroxy-3-methyloct-1-enyl)-3-methyl-5-oxocyclopent-3-enyl)hept-5-enoicAcid

Step A: Preparation of(3aR,4R,5R,6aS)-2-oxo-4-((E)-3-oxooct-1-enyl)hexahydro-2H-cyclopenta[b]furan-5-ylBenzoate

A reactor equipped with a mechanical stirrer, under nitrogen, wascharged with(3aR,4R,5R,6aS)-4-formyl-2-oxohexahydro-2H-cyclopenta[b]furan-5-ylbenzoate (Corey lactone aldehyde benzoate, Cayman Chemical Catalog No.70030, 99.2 g, 0.362 mol) in DCM and lithium chloride (1 molarequivalent) dissolved in THF. Some lithium chloride precipitated fromsolution when the THF and DCM solutions were mixed. Dimethyl2-oxoheptylphosphonate (1 molar equivalent) was subsequently added tothe reactor NEAT and the reagent was rinsed down into the reactor withDCM. The mixture was stirred under nitrogen and cooled to −20° C. Thelithium chloride precipitate dissolved as the stirring and coolingcontinued. After stirring for 2.5 hours, triethylamine (1 molarequivalent) was added NEAT via addition funnel and the temperature wasmaintained at −5° C. with stirring for 19 hours. The reaction mixturewas warmed to 0° C. and treated with 5% aqueous citric acid. The layerswere separated and the organic layer was dried over magnesium sulfate,filtered, and concentrated under reduced pressure. The crude product waspurified on silica gel, eluted with hexanes-ethyl acetates (1:1) toafford the title intermediate.

Step B: Preparation of(3aR,4R,5R,6aS)-4-((E)-3-hydroxy-3-methyloct-1-enyl)-2-oxohexahydro-2H-cyclopenta[b]furan-5-ylBenzoate

To a stirring mixture consisting of(3aR,4R,5R,6aS)-2-oxo-4-((E)-3-oxooct-1-enyl)hexahydro-2H-cyclopenta[b]furan-5-ylbenzoate (limiting reagent, prepared in Step A) in THF (0.1 M) under anitrogen atmosphere cooled to −78° C. was added methyl magnesium bromide(1 M solution in THF, 1 molar equivalent) dropwise. The reaction mixturewas stirred at −78° C. until reaction progress stopped. Upon completion,brine was added to the crude reaction mixture and the product wasextracted with ethyl acetate. The combined organic layers were driedover sodium sulfate, filtered, and evaporated. The product was purifiedby flash chromatography on regular silica gel, eluted with hexanes-ethylacetate to afford the title intermediate.

Step C: Preparation of(3aR,4R,5R,6aS)-4-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-2-oxohexahydro-2H-cyclopenta[b]furan-5-ylBenzoate

To a stirring mixture consisting of(3aR,4R,5R,6aS)-4-((E)-3-hydroxy-3-methyloct-1-enyl)-2-oxohexahydro-2H-cyclopenta[b]furan-5-ylbenzoate (limiting reagent, prepared in Step B) and imidazole (1.1.molar equivalents) in DMF (5 M in limiting reagent) cooled to 0° C.under a nitrogen atmosphere was slowly added a solution consisting ofTBDPSCl (1.1 molar equivalent) in DMF. Upon completion of the reaction,as judged by TLC, the reaction mixture was diluted with ethyl acetateand washed with water and brine. The organic layer was dried over sodiumsulfate, filtered, and evaporated. The crude product was purified byflash chromatography on regular silica gel eluted with hexanes-ethylacetate to afford the title intermediate.

Step D: Preparation of(3aR,4R,5R,6aS)-4-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-5-hydroxyhexahydro-2H-cyclopenta[b]furan-2-one

To a stirring mixture consisting of(3aR,4R,5R,6aS)-4-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-2-oxohexahydro-2H-cyclopenta[b]furan-5-ylbenzoate (limiting reagent, prepared in Step C) in methanol (0.2 M) wasadded potassium carbonate (0.6 molar equivalent). The reaction mixturewas stirred at room temperature and the progress was monitored by TLCevery 30 minutes. After complete consumption of starting material, thereaction mixture was acidified with 5% KHSO₄ and diluted with brine. Theproduct was extracted with ethyl acetate twice. The combined organiclayers were dried over sodium sulfate, filtered, and evaporated. Thecrude product was purified by flash chromatography on regular silica geleluted with hexanes-ethyl acetate (1:1) to afford the titleintermediate.

Step E: Preparation of(3aR,4R,5R,6aS)-4-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-5-(tetrahydro-2H-pyran-2-yloxy)hexahydro-2H-cyclopenta[b]furan-2-one

To a stirring mixture consisting of(3aR,4R,5R,6aS)-4-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-5-hydroxyhexahydro-2H-cyclopenta[b]furan-2-one(limiting reagent, prepared in Step D) in DCM (0.1 M) under a nitrogenatmosphere was added dihydropyran (1.1 molar equivalent) followed by acatalytic amount of p-toluenesulfonic acid. The reaction mixture wasstirred at room temperature under a nitrogen atmosphere and the reactionprogress was monitored by TLC. Upon completion, brine was added to thereaction mixture and the layers were separated. The organic phase wasdried over sodium sulfate, filtered, and the solvent was evaporated. Thecrude product was purified by flash chromatography on regular silica geleluted with hexanes-ethyl acetate to afford the title intermediate.

Step F: Preparation of(3aR,4R,5R,6aS)-4-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-5-(tetrahydro-2H-pyran-2-yloxy)hexahydro-2H-cyclopenta[b]furan-2-ol

A stirring mixture consisting of(3aR,4R,5R,6aS)-4-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-5-(tetrahydro-2H-pyran-2-yloxy)hexahydro-2H-cyclopenta[b]furan-2-one(limiting reagent, prepared in Step E) in anhydrous THF (0.5 M) under anitrogen atmosphere was cooled to −78° C. A solution consisting ofDIBAL-H (1 M in toluene, 2 molar equivalents) was added to the reactionmixture dropwise and stirred for 3 hours. Ethyl acetate (20 mL) wasadded and the mixture was stirred for an additional 5 minutes. Themixture was subsequently treated with 30% aqueous K, Na tartrate andstirred vigorously overnight. The layers were separated and the organicphase was dried, filtered and concentrated under reduced pressure. Thecrude product was purified by flash chromatography on regular silica geleluted with hexanes-ethyl acetate to afford the title intermediate.

Step G: Preparation of(Z)-7-((1R,2R,3R,5S)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-5-hydroxy-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoicAcid

To a white suspension consisting of 4-carboxybutyltriphenylphosphoniumbromide (3.5 molar equivalents) in anhydrous THF under nitrogenatmosphere was added dropwise a solution consisting of 1 M potassiumtert-butoxide (7 molar equivalents) in THF. The reaction mixture becamebright red over the course of the addition and was stirred for 30minutes at room temperature and subsequently cooled to −15° C. with aice/NaCl bath. The lactol(3aR,4R,5R,6aS)-4-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-5-(tetrahydro-2H-pyran-2-yloxy)hexahydro-2H-cyclopenta[b]furan-2-ol(limiting reagent, prepared in Step F) was dissolved in THF and addeddropwise to the reaction mixture containing the ylide. The reactionmixture became lighter orange in color and was stirred for 2 hours at−15° C. and was subsequently allowed to warm to room temperature andstir overnight. The reaction mixture became dark red and TLC analysisindicated no remaining starting material. The reaction mixture wasacidified with 5% KHSO₄, diluted with brine (250 mL), and extracted withethyl acetate (200 mL). The aqueous layer was extracted with anotherportion of ethyl acetate (50 mL) and the combined organic extracts werewashed twice with brine (2×250 mL), dried over sodium sulfate, andevaporated. The crude product was purified by flash chromatography onregular silica gel using hexanes-ethyl acetate with 0.4% acetic acid aseluent to afford the title intermediate.

Step H: Preparation of (Z)-methyl7-((1R,2R,3R,5S)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-5-hydroxy-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoate

A stirring mixture consisting of(Z)-7-((1R,2R,3R,5S)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-5-hydroxy-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoicacid (prepared in Step G) in diethyl ether (0.1 M) was cooled to 0° C.under a nitrogen atmosphere. Diazomethane (freshly prepared solution indiethyl ether) was added to the stirring mixture until a light-yellowcolor persisted. The completion of the reaction was confirmed by theabsence of starting material as judged by TLC. Upon completion, thesolvents were evaporated and the product was purified by flashchromatography using hexanes-ethyl acetate as eluent to afford the titleintermediate.

Step I: Preparation of (Z)-methyl7-((1R,2R,3R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoate

To a stirring mixture consisting of (Z)-methyl7-((1R,2R,3R,5S)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-5-hydroxy-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoate(limiting reagent, prepared in Step H) in DMF (5 M) and imidazole (1.1molar equivalents) cooled to 0° C. under a nitrogen atmosphere wasslowly added a solution consisting of TBDPSCl (1.1 molar equivalent) inDMF. Upon completion of the reaction, as judged by TLC, the reactionmixture was diluted with ethyl acetate and was washed with water andbrine. The organic layer was dried over sodium sulfate, filtered, andevaporated. The crude product was purified by flash chromatography onregular silica gel eluted with hexanes-ethyl acetate to afford the titleintermediate.

Step J: Preparation of (Z)-methyl7-((1R,2R,3R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-3-hydroxycyclopentyl)hept-5-enoate

A mixture consisting of (Z)-methyl7-((1R,2R,3R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3-(tert-butydiphenylsilyloxy)-3-methyloct-1-enyl)-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoate(prepared in Step I) in 4:2:1 acetic acid-water-THF (0.5 M) was stirredfor several days at room temperature until the reaction was complete, asjudged by TLC. The solvents were removed by evaporation and the crudeproduct was purified by flash chromatography on regular silica gel usinghexanes-ethyl acetate with 0.4% acetic acid as eluent to afford thetitle intermediate.

Step K: Preparation of (Z)-methyl7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-3-oxocyclopentyl)hept-5-enoate

To a stirring mixture consisting of (Z)-methyl7-((1R,2R,3R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-3-hydroxycyclopentyl)hept-5-enoate(limiting reagent, prepared in Step J) in acetone (0.1 M) cooled to −25°C. was added Jones reagent (1 molar equivalent) dropwise. Uponcompletion, as judged by TLC, the reaction was quenched with isopropylalcohol and the crude reaction mixture was diluted with ethyl acetate,washed three times with brine, and dried over magnesium sulfate. Afterfiltration and solvent evaporation the product was purified by flashchromatography using hexanes-ethyl acetate as eluent to afford the titleintermediate.

Step L: Preparation of (Z)-methyl7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-3-methylenecyclopentyl)hept-5-enoate

A stirring mixture consisting of (Z)-methyl7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-3-oxocyclopentyl)hept-5-enoate(limiting reagent, prepared in Step K) in DCM (0.05 M) under a nitrogenatmosphere was cooled to 0° C. A zinc methylenedibromide titaniumtetrachloride solution was prepared by combining stirring zinc dust (2.3g) in THF (40 mL) with methylene dibromide (0.81 mL) at −40° C. under anitrogen atmosphere. To the suspension was slowly added TiCl₄ (0.92 mL).Portions (2 mL) of zinc methylenedibromide titanium tetrachloridesolution were added to the stirring mixture containing the limitingreagent until the reaction was complete as judged by TLC. Uponcompletion, the reaction mixture was diluted with ethyl acetate andfiltered twice through a bed of Celite. The filtrate was washed with asaturated aqueous solution of sodium bicarbonate and subsequently with a50% aqueous solution of brine. The organic phase was dried over sodiumsulfate, filtered, and the solvent was evaporated. The crude product waspurified by flash chromatography on regular silica gel eluted withhexanes-ethyl acetate to afford the title intermediate.

Step M: Preparation of(Z)-7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-3-methylenecyclopentyl)hept-5-enoicAcid

A mixture consisting of (Z)-methyl7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-3-methylenecyclopentyl)hept-5-enoate(limiting reagent, prepared in Step L) in a 3:1 solution of methanol and1 N LiOH (0.01 M) was stirred at 4° C. Upon completion of the reaction,as judged by TLC, the reaction mixture was diluted with ethyl acetate,washed with 5% KHSO₄, and brine. The organic phase was dried over sodiumsulfate, filtered, and the solvent was evaporated. The crude product waspurified by flash chromatography on regular silica gel eluted withhexanes-ethyl acetate to afford the title intermediate.

Step N: Preparation of(Z)-7-((1R,2R,5S)-5-hydroxy-2-((E)-3-hydroxy-3-methyloct-1-enyl)-3-methylenecyclopentyl)hept-5-enoicAcid

To a stirring mixture consisting of(Z)-7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3-(tert-butydiphenylsilyloxy)-3-methyloct-1-enyl)-3-methylenecyclopentyl)hept-5-enoicacid (limiting reagent, prepared in Step M) in THF (0.3 M) under anitrogen atmosphere was added a solution of TBAF (1.2 molar equivalents;1 M in THF) and the reaction mixture was stirred at room temperature.Upon completion, as judged by TLC, water was added and most of the THFwas removed under reduced pressure. The remaining aqueous solution wasextracted with ethyl acetate and was washed with water and brine. Theorganic phase was dried over sodium sulfate, filtered, and the solventwas evaporated. The crude product was purified by flash chromatographyon regular silica gel eluted with hexanes-ethyl acetate with 0.4% aceticacid to afford the title compound.

Step O: Preparation of(Z)-7-((1R,2R)-2-((E)-3-hydroxy-3-methyloct-1-enyl)-3-methyl-5-oxocyclopent-3-enyl)hept-5-enoicAcid

To a stirring mixture consisting of(Z)-7-((1R,2R,5S)-5-hydroxy-2-((E)-3-hydroxy-3-methyloct-1-enyl)-3-methylenecyclopentyl)hept-5-enoicacid (limiting reagent, prepared in Step N) in acetone (0.1 M) cooled to−25° C. was added Jones reagent (1 molar equivalent) dropwise. Uponcompletion, as judged by TLC, the reaction was quenched with isopropylalcohol and the crude reaction mixture was diluted with ethyl acetate,washed three times with brine, and dried over magnesium sulfate. Afterfiltration and solvent evaporation the product was purified by flashchromatography using hexanes-ethyl acetate as eluent to afford anepimeric mixture of the title compound. The 15-hydroxy and 15-methylepimers may be resolved or used as an epimeric mixture.

Example 2 Preparation of(Z)-7-((1R,2R)-2-((E)-3,3-difluorooct-1-enyl)-3-methyl-5-oxocyclopent-3-enyl)hept-5-enoicAcid

Step A: Preparation of(3aR,4R,5R,6aS)-4-((E)-3,3-difluorooct-1-enyl)-2-oxohexahydro-2H-cyclopenta[b]furan-5-ylBenzoate

To a stirring mixture consisting of(3aR,4R,5R,6aS)-2-oxo-4-((E)-3-oxooct-1-enyl)hexahydro-2H-cyclopenta[b]furan-5-ylbenzoate (limiting reagent, prepared in Example 1, Step A) and acatalytic amount of ethanol (25 mole %) in anhydrous DCM (0.5 M inlimiting reagent) cooled to 0° C. under nitrogen a atmosphere is slowlyadded DAST (5 molar equivalents). The reaction mixture is allowed toslowly warm to room temperature overnight. Stirring is continued forseveral days until the reaction is complete as judged by TLC. Uponcompletion the reaction is cooled to 0° C. and quenched by the slowaddition of a saturated aqueous solution of sodium bicarbonate. Thelayers are separated and the aqueous phase is extracted with ethylacetate. The organic layers are combined and dried over magnesiumsulfate. The solvents are evaporated and the crude material is purifiedon regular silica gel eluted with hexanes-ethyl acetate to afford thetitle intermediate.

Step B: Preparation of(3aR,4R,5R,6aS)-4-((E)-3,3-difluorooct-1-enyl)-5-hydroxyhexahydro-2H-cyclopenta[b]furan-2-one

The title intermediate may be prepared from(3aR,4R,5R,6aS)-4-((E)-3,3-difluorooct-1-enyl)-2-oxohexahydro-2H-cyclopenta[b]furan-5-ylbenzoate (prepared in Step A) using a procedure described in Example 1,Step D for the removal of the benzoate group.

Step C: Preparation of(3aR,4R,5R,6aS)-4-((E)-3,3-difluorooct-1-enyl)-5-(tetrahydro-2H-pyran-2-yloxy)hexahydro-2H-cyclopenta[b]furan-2-one

The title intermediate may be prepared from(3aR,4R,5R,6aS)-4-((E)-3,3-difluorooct-1-enyl)-5-hydroxyhexahydro-2H-cyclopenta[b]furan-2-one(prepared in Step B) using the THP-protection procedure described inExample 1, Step E.

Step D: Preparation of(3aR,4R,5R,6aS)-4-((E)-3,3-difluorooct-1-enyl)-5-(tetrahydro-2H-pyran-2-yloxy)hexahydro-2H-cyclopenta[b]furan-2-ol

The title intermediate may be prepared from(3aR,4R,5R,6aS)-4-((E)-3,3-difluorooct-1-enyl)-5-(tetrahydro-2H-pyran-2-yloxy)hexahydro-2H-cyclopenta[b]furan-2-one(prepared in Step C) using the procedure described in Example 1, Step F.

Step E:(Z)-7-((1R,2R,3R,5S)-2-((E)-3,3-difluorooct-1-enyl)-5-hydroxy-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoicAcid

The title intermediate may be prepared from the lactol(3aR,4R,5R,6aS)-4-((E)-3,3-difluorooct-1-enyl)-5-(tetrahydro-2H-pyran-2-yloxy)hexahydro-2H-cyclopenta[b]furan-2-ol(prepared in Step D) using the procedure described in Example 1, Step G.

Step F: Preparation of (Z)-methyl7-((1R,2R,3R,5S)-2-((E)-3,3-difluorooct-1-enyl)-5-hydroxy-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoate

The title intermediate may be prepared from(Z)-7-((1R,2R,3R,5S)-2-((E)-3,3-difluorooct-1-enyl)-5-hydroxy-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoicacid (prepared in Step E) using the diazomethane procedure described inExample 1, Step H.

Step G: Preparation of (Z)-methyl7-((1R,2R,3R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluorooct-1-enyl)-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoate

The title intermediate may be prepared from (Z)-methyl7-((1R,2R,3R,5S)-2-((E)-3,3-difluorooct-1-enyl)-5-hydroxy-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoate(prepared in Step F) using the silylation procedure described in Example1, Step I.

Step H: Preparation of (Z)-methyl7-((1R,2R,3R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluorooct-1-enyl)-3-hydroxycyclopentyl)hept-5-enoate

The title intermediate may be prepared form (Z)-methyl7-((1R,2R,3R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluorooct-1-enyl)-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoate(prepared in Step G) using the THP-deprotection procedure described inExample 1, Step J.

Step I: Preparation of (Z)-methyl7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluorooct-1-enyl)-3-oxocyclopentyl)hept-5-enoate

The title intermediate may be prepared from (Z)-methyl7-((1R,2R,3R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluorooct-1-enyl)-3-hydroxycyclopentyl)hept-5-enoate(prepared in Step H) using the Jones oxidation procedure described inExample 1, Step K.

Step J: Preparation of (Z)-methyl7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluorooct-1-enyl)-3-methylenecyclopentyl)hept-5-enoate

The title intermediate may be prepared from (Z)-methyl7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluorooct-1-enyl)-3-oxocyclopentyl)hept-5-enoate(prepared in Step I) using the procedure described in Example 1, Step L.

Step K: Preparation of(Z)-7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluorooct-1-enyl)-3-methylenecyclopentyl)hept-5-enoicAcid

The title intermediate may be prepared from (Z)-methyl7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluorooct-1-enyl)-3-methylenecyclopentyl)hept-5-enoate(prepared in Step J) using the ester hydrolysis procedure described inExample 1, Step M.

Step L: Preparation of(Z)-7-((1R,2R,5S)-2-((E)-3,3-difluorooct-1-enyl)-5-hydroxy-3-methylenecyclopentyl)hept-5-enoicAcid

The title compound may be prepared from(Z)-7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluorooct-1-enyl)-3-methylenecyclopentyl)hept-5-enoicacid (prepared in Step K) using the silyl-deprotection proceduredescribed in Example 1, Step N.

Step M: Preparation of(Z)-7-((1R,2R)-2-((E)-3,3-difluorooct-1-enyl)-3-methyl-5-oxocyclopent-3-enyl)hept-5-enoicAcid

The title compound may be prepared from(Z)-7-((1R,2R,5S)-2-((E)-3,3-difluorooct-1-enyl)-5-hydroxy-3-methylenecyclopentyl)hept-5-enoicacid (prepared in Step L) using the Jones oxidation procedure describedin Example 1, Step O.

Example 3 Preparation of(Z)-7-((1R,2R,3R)-2-((E)-3,3-difluoro-4-hydroxyoct-1-enyl)-3-hydroxy-5-oxocyclopentyl)hept-5-enoicAcid

Step A: Preparation of dimethyl2-oxo-3-(tetrahydro-2H-pyran-2-yloxy)heptylphosphonate

To a stirring mixture consisting of methyl 2-hydroxyhexanoate (limitingreagent, available from Sinochemexper) in DCM (0.1 M) under a nitrogenatmosphere is added dihydropyran (1.1 molar equivalents) followed by acatalytic amount of p-toluenesulfonic acid. The reaction mixture isstirred at room temperature and the reaction progress is monitored byTLC. Upon completion, brine is added to the reaction mixture and thelayers are separated. The organic phase is dried over sodium sulfate,filtered, and the solvent is evaporated. The crude product is purifiedby flash chromatography on regular silica gel eluted with hexanes-ethylacetate to give methyl 2-(tetrahydro-2H-pyran-2-yloxy)hexanoate. Methyl2-(tetrahydro-2H-pyran-2-yloxy)hexanoate is converted to the titleintermediate by treating with diethyl methyphosphonate as previouslydescribed in the Journal of Organic Chemistry, 73 (12), 200 8,4568-4574.

Step B: Preparation of(3aR,4R,5R,6aS)-2-oxo-4-((E)-3-oxo-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)hexahydro-2H-cyclopenta[b]furan-5-ylBenzoate

A reactor equipped with a mechanical stirrer purged with nitrogen gas ischarged with(3aR,4R,5R,6aS)-4-formyl-2-oxohexahydro-2H-cyclopenta[b]furan-5-ylbenzoate (99.2 g, 0.362 mol) dissolved in DCM and lithium chloride(0.362 mol) dissolved in THF. Some lithium chloride precipitates fromsolution when the THF and DCM solutions are mixed. Dimethyl2-oxo-3-(tetrahydro-2H-pyran-2-yloxy)heptylphosphonate (0.362 mol,prepared in Step A) is added NEAT and rinsed into the reaction vesselwith DCM. The mixture is stirred under nitrogen and cooled to −20° C.and the lithium chloride precipitate dissolves. After stirring for 2.5hours, triethylamine (0.362 mol) is added NEAT via addition funnel andthe temperature is maintained at −5° C. and stirring is continued for 19hours. The temperature is adjusted to 0° C. and the reaction mixturetreated with 5% aqueous citric acid. The layers are separated and theorganic layer is dried over magnesium sulfate, filtered, and evaporated.The crude product is purified on silica gel. Elution with hexanes-ethylacetate affords the title intermediate.

Step C: Preparation of(3aR,4R,5R,6aS)-4-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-2-oxohexahydro-2H-cyclopenta[b]furan-5-ylBenzoate

The title intermediate may be prepared from(3aR,4R,5R,6aS)-4-((E)-4-(tert-butyldimethylsilyloxy)-3-oxooct-1-enyl)-2-oxohexahydro-2H-cyclopenta[b]furan-5-ylbenzoate (prepared in Step B) using the DAST fluorination proceduredescribed in Example 2, Step A.

Step D: Preparation of(3aR,4R,5R,6aS)-4-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxyhexahydro-2H-cyclopenta[b]furan-2-one

The title intermediate may be prepared from(3aR,4R,5R,6aS)-4-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-2-oxohexahydro-2H-cyclopenta[b]furan-5-ylbenzoate (prepared in Step C) using a procedure described in Example 1,Step D for the removal of the benzoate group.

Step E: Preparation of(3aR,4R,5R,6aS)-4-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)hexahydro-2H-cyclopenta[b]furan-2,5-diol

The title intermediate may be prepared from(3aR,4R,5R,6aS)-4-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxyhexahydro-2H-cyclopenta[b]furan-2-one(prepared in Step D) using the reduction procedure described in Example1, Step F.

Step F: Preparation of(Z)-7-((1R,2R,3R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3,5-dihydroxycyclopentyl)hept-5-enoicAcid

The title intermediate may be prepared from(3aR,4R,5R,6aS)-4-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)hexahydro-2H-cyclopenta[b]furan-2,5-diol(prepared in Step E) using the procedure described in Example 1, Step G.

Step G: Preparation of(Z)-7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-oxocyclopentylhept-5-enoic acid and(Z)-7-((1R,2R,3R)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3-hydroxy-5-oxocyclopentyl)hept-5-enoicAcid

To a stirring mixture consisting of(Z)-7-((1R,2R,3R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3,5-dihydroxycyclopentyl)hept-5-enoicacid (limiting reagent, prepared in Step F) in acetone (0.1 M) cooled to−25° C. is added Jones reagent (1 molar equivalent) dropwise. Uponcompletion, as judged by TLC, the reaction is quenched with isopropylalcohol and the crude reaction mixture is diluted with ethyl acetate,washed three times with brine, and dried over magnesium sulfate. Afterfiltration and solvent evaporation, the product mixture is separated byflash chromatography using ethyl acetate-hexane as eluent to afford theseparated regioisomers(Z)-7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-oxocyclopentyl)hept-5-enoicacid and(Z)-7-((1R,2R,3R)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3-hydroxy-5-oxocyclopentyl)hept-5-enoicacid.

Step H: Preparation of(Z)-7-((1R,2R,3R)-2-((E)-3,3-difluoro-4-hydroxyoct-1-enyl)-3-hydroxy-5-oxocyclopentyl)hept-5-enoicAcid

The title compound may be prepared from(Z)-7-((1R,2R,3R)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3-hydroxy-5-oxocyclopentyl)hept-5-enoicacid (prepared in Step G) using the THP-deprotection procedure describedin Example 1, Step J.

Example 4 Preparation of(Z)-7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-hydroxyoct-1-enyl)-5-hydroxy-3-oxocyclopentyl)hept-5-enoicAcid

The title compound may be prepared from(Z)-7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-oxocyclopentyl)hept-5-enoicacid (prepared in Example 3, Step G) using the THP-deprotectionprocedure described in Example 1, Step J.

Example 5 Preparation of(Z)-7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-hydroxyoct-1-enyl)-5-hydroxy-3-methylenecyclopentyl)hept-5-enoicAcid

Step A: Preparation of (Z)-methyl7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-oxocyclopentyl)hept-5-enoate

The title intermediate may be prepared from(Z)-7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-oxocyclopentyl)hept-5-enoicacid (prepared in Example 3, Step G) using the diazomethane proceduredescribed in Example 1, Step H.

Step B: Preparation of (Z)-methyl7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3-oxocyclopentyl)hept-5-enoate

The title intermediate may be prepared from (Z)-methyl7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-oxocyclopentyl)hept-5-enoate(prepared in Step A) using the silylation procedure described in Example1, Step I.

Step C: Preparation of (Z)-methyl7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3-methylenecyclopentyl)hept-5-enoate

The title intermediate may be prepared from (Z)-methyl7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3-oxocyclopentyl)hept-5-enoate(prepared in Step B) using the procedure described in Example 1, Step L.

Step D: Preparation of (Z)-methyl7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-methylenecyclopentyl)hept-5-enoate

The title intermediate may be prepared from (Z)-methyl7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3-methylenecyclopentyl)hept-5-enoate(prepared in Step C) using the TBDPS-deprotection procedure described inExample 1, Step N.

Step E: Preparation of(Z)-7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-methylenecyclopentyl)hept-5-enoicAcid

The title intermediate may be prepared from (Z)-methyl7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-methylenecyclopentyl)hept-5-enoate(prepared in Step D) using the ester hydrolysis procedure described inExample 1, Step M.

Step F: Preparation of(Z)-7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-hydroxyoct-1-enyl)-5-hydroxy-3-methylenecyclopentyl)hept-5-enoicAcid

The title compound may be prepared from(Z)-7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-methylenecyclopentyl)hept-5-enoicacid (prepared in Step E) using the THP-deprotection procedure describedin Example 1, Step J.

Example 6 Preparation of(Z)-7-((1R,2R)-2-((E)-3,3-difluoro-4-hydroxyoct-1-enyl)-3-methyl-5-oxocyclopent-3-enyl)hept-5-enoicAcid

Step A: Preparation of(Z)-7-((1R,2R)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3-methyl-5-oxocyclopent-3-enyl)hept-5-enoicAcid

The title intermediate may be prepared from(Z)-7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-methylenecyclopentyl)hept-5-enoicacid (prepared in Example 5, Step E) using the Jones oxidation proceduredescribed in Example 1, Step O.

Step B: Preparation of(Z)-7-((1R,2R)-2-((E)-3,3-difluoro-4-hydroxyoct-1-enyl)-3-methyl-5-oxocyclopent-3-enyl)hept-5-enoicAcid

The title compound may be prepared from(Z)-7-((1R,2R)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3-methyl-5-oxocyclopent-3-enyl)hept-5-enoicacid (prepared in Step A) using the THP-deprotection procedure describedin Example 1, Step J.

EP Receptor Binding and Agonism

The ability of compounds to bind the EP receptors and their selectivityfor each receptor can be demonstrated in radioligand competitiondisplacement binding experiments using the cell lines described abovewhich stably overexpress the human EP receptors. The ability ofcompounds to activate the receptors can be demonstrated in secondmessenger functional assays, measuring changes in intracellular calciumfor EP₁ and changes in cAMP formation for EP₂, EP₃ and EP₄.

Test Details

Binding Ability to Human EP Receptors

Membranes are prepared from cells stably transfected with human EPreceptor DNA. In brief, cells are cultured to confluence, scraped fromculture flasks and centrifuged to pellet (800×g, 5 minutes, 4° C.).Cells are washed twice with ice-cold homogenization buffer containing 10mM Tris-HCl, 1 mM EDTA, 250 mM sucrose, 1 mM PMSF, 300 μM indomethacin,pH 7.4, homogenized by sonication and centrifuged as before. Thesupernatant is stored on ice; the pellets are rehomogenized and respun.Supernatants are pooled and centrifuged at 100,000×g for 10 minutes at4° C. The resultant membrane pellet is stored at −80° C. until use.

For assays, membranes from cells expressing human EP₁, EP₂, EP₃ or EP₄receptors are added to assay buffer (10 mM MES, pH 6.0, 10 mM MgCl₂, 1mM EDTA, 3 μM indomethacin) containing 5 nM [³H]-PGE₂ (GE Healthcare)and 0.1 to 10,000 nM concentrations of compounds to be tested.Incubations are performed at suitable temperatures and times to allowequilibration to be reached. Non-specific binding is determined in thepresence of 10 μM PGE₂. Reactions are terminated by the addition ofice-cold buffer followed by rapid filtration through Whatman GF/Bfilters. The filters are dried after washing, and membrane-boundradioactivity is quantified by scintillation counting.

The affinity or pK_(i) of each compound for each receptor is calculatedfrom the concentration causing 50% radioligand displacement (IC₅₀) usingthe Cheng-Prosoff equation:

K _(i) =IC ₅₀/[1+(radioligand concentration/radioligand K _(d))]

Functional Assays: Effect of Compounds on Second Messenger Generation

The following sections describe in vitro assays to determine the effectof compounds on calcium mobilization, and on the induction or inhibitionof cAMP generation, that is, to determine the functional efficacy ofcompounds at the EP₁ (calcium mobilization), EP₂ (induction of cAMP),EP₃ (inhibition of forskolin-induced cAMP) or EP₄ (induction of cAMP)receptor.

EP₁ Receptor Agonism Assay (Intracellular Calcium Assay)

Functional Assay #1AGi

To test the ability of compounds to activate the EP₁ receptor, calciummobilization experiments are performed. Cells expressing the EP₁receptor are plated in clear-bottom black 96-well plates in normalgrowth medium and grown to confluence. When the cells have reachedconfluence, the culture medium is replaced with 50 μl of Fluo-4 NW dyemix (Invitrogen) that is dissolved in Hank's balanced salt solutioncontaining 20 mM HEPES, pH 7.4 and 2.5 mM probenecid. Experiments areinitiated by the addition of 50 μl/well of vehicle or compound to betested diluted in this same buffer. Plates are incubated for 30 minutesat 37° C. and then at room temperature for an additional 30 minutes.Calcium fluorescence is measured using an Analyst AD (Molecular Devices)with an excitation wavelength of 485 nm, emission wavelength of 560 nm,and emission cutoff of 505 nm. Responses are quantified as peakfluorescence intensity minus basal fluorescence intensity.

Alternative EP₁ Receptor Agonism Assay

Functional Assay #1AGii

(Cerep, Catalog reference 722-55a; UNGRIN, M. D., SINGH L. M. R.,STOCCO, R., SAS, D. E. and ABRAMOVITZ, M. (1999), An automated aequorinluminescence-based functional calcium assay for G-Protein-CoupledReceptors. Analytical Biochem., 272, 34.)

Evaluation of the agonist activity of compounds at the human EP₁receptor in transfected HEK-293 cells, determined by measuring theireffect on cytosolic Ca²⁺ ion mobilization using a fluorimetric detectionmethod.

The cells are suspended in DMEM buffer (Invitrogen), then distributed inmicroplates at a density of 3·10⁴ cells/well. The fluorescent probe(Fluo4 NW, Invitrogen) mixed with probenicid in HBSS buffer (Invitrogen)complemented with 20 mM Hepes (Invitrogen) (pH 7.4) is then added intoeach well and equilibrated with the cells for 30 minutes at 37° C. then30 minutes at 22° C. Thereafter, the assay plates are positioned in amicroplate reader (CellLux, PerkinElmer) which is used for the additionof the test compound, reference agonist or HBSS buffer (basal control),and the measurements of changes in fluorescence intensity which variesproportionally to the free cytosolic Ca²⁺ ion concentration. Forstimulated control measurements, PGE₂ at 100 nM is added in separateassay wells.

The results are expressed as a percent of the control response to 100 nMPGE₂. The standard reference agonist is PGE₂, which is tested in eachexperiment at several concentrations to generate aconcentration-response curve from which its EC₅₀ value is calculated.

EP₁ Receptor Antagonism Assay

Functional Assay #1ANT

(Cerep, Catalog Reference 722-55B; Ungrin, M. D., et al., Ibid.)

Evaluation of the antagonist activity of compounds at the human EP₁receptor in transfected HEK-293 cells, determined by measuring theireffect on agonist-induced cytosolic Ca²⁺ ion mobilization using afluorimetric detection method.

The cells are suspended in DMEM buffer (Invitrogen), then distributed inmicroplates at a density of 3·10⁴ cells/well. The fluorescent probe(Fluo4 NW, Invitrogen) mixed with probenicid in HBSS buffer (Invitrogen)complemented with 20 mM Hepes (Invitrogen) (pH 7.4) is then added intoeach well and equilibrated with the cells for 30 minutes at 37° C. then30 minutes at 22° C. Thereafter, the assay plates are positioned in amicroplate reader (CellLux, PerkinElmer) which is used for the additionof the test compound, reference antagonist or HBSS buffer (basalcontrol), then 5 minutes later 3 nM PGE₂, and the measurements ofchanges in fluorescence intensity which varies proportionally to thefree cytosolic Ca²⁺ ion concentration. The results are expressed as apercent inhibition of the control response to 3 nM PGE₂. The standardreference antagonist is SC 51322, which is tested in each experiment atseveral concentrations to generate a concentration-response curve fromwhich its IC₅₀ value is calculated.

EP₂ and EP₄ Receptor Agonism Assay (Cyclic AMP Induction Assay)

Functional Assay #2AGi and Functional Assay #4AGi, Respectively

To test the ability of compounds to activate the EP₂ and EP₄ receptors,accumulation of cAMP following treatment with these compounds ismeasured. Cells expressing the EP₂ or EP₄ receptor are plated in 24-wellplates in normal growth medium and grown to confluence. When the cellshave reached confluence, the medium is replaced with 450 l of serum-freemedium containing 0.25 mM IBMX and 20 μM indomethacin. Cells areincubated in this medium for one hour. Fifty microliters of this samebuffer containing various concentrations of PGE₂ or compounds to betested is subsequently added to the cells and the cells are incubatedfor fifteen to thirty minutes to allow the accumulation of cAMP.Reactions are terminated by the addition of 500 μl of 10% TCA. cAMPmeasurements of the cell lysates are performed using Cayman Chemical'scommercially available cAMP EIA Kit following the instructions providedin the kit booklet.

Alternative EP₂ Receptor Agonism Assay

Functional Assay #2AGii

(Cerep, Catalog reference 758-54a; Wilson, R. J., Rhodes, S. A., Wood,R. L., Shield, V. J., Noel, L. S., Gray, D. W. and Giles H. (2004),Functional pharmacology of human prostanoid EP₂ and EP4 receptors, Eur.J. Pharmacol., 501, 49.)

Evaluation of the agonist activity of compounds at the human EP₂receptor in transfected CHO cells, determined by measuring their effectson cAMP production using the HTRF detection method.

The cells are suspended in HBSS buffer (Invitrogen) complemented withHEPES 20 mM (pH 7.4) and 500 μM IBMX, then distributed in microplates ata density of 10⁴ cells/well and incubated for 30 minutes at 37° C. inthe absence (control) or presence of the test compound or the referenceagonist. For stimulated control measurements, separate assay wellscontain 10 μM PGE₂. Following incubation, the cells are lysed and thefluorescence acceptor (D2-labeled cAMP) and fluorescence donor(anti-cAMP antibody labeled with europium cryptate) are added. After 60minutes at room temperature, the fluorescence transfer is measured atλex=337 nm and λem=620 and 665 nm using a microplate reader (Rubystar,BMG). The cAMP concentration is determined by dividing the signalmeasured at 665 nm by that measured at 620 nm (ratio). The results areexpressed as a percent of the control response to 10 μM PGE₂. Thestandard reference agonist is PGE₂, which is tested in each experimentat several concentrations to generate a concentration-response curvefrom which its EC₅₀ value is calculated.

EP₂ Receptor Antagonism Assay

Functional Assay #2ANT

(Cerep, Catalog Reference 758-54B; Wilson, R. J., et al., Ibid.)

Evaluation of the antagonist activity of compounds at the human EP₂receptor in transfected CHO cells, determined by measuring their effectson agonist-induced cAMP production using the HTRF detection method.

The cells are suspended in HBSS buffer (Invitrogen) complemented withHEPES 20 mM (pH 7.4) and 500 μM IBMX, then distributed in microplates ata density of 10⁴ cells/well and preincubated for 5 minutes at roomtemperature in the absence (control) or presence of the test compound orthe reference antagonist. Thereafter, the reference agonist PGE₂ isadded at a final concentration of 300 nM. For basal controlmeasurements, separate assay wells do not contain PGE₂. Following 30minutes incubation at 37° C., the cells are lysed and the fluorescenceacceptor (D2-labeled cAMP) and fluorescence donor (anti-cAMP antibodylabeled with europium cryptate) are added. After 60 minutes at roomtemperature, the fluorescence transfer is measured at λex=337 nm andλem=620 and 665 nm using a microplate reader (Rubystar, BMG). The cAMPconcentration is determined by dividing the signal measured at 665 nm bythat measured at 620 nm (ratio). The results are expressed as a percentinhibition of the control response to 300 nM PGE₂. The standardreference antagonist is AH 6809, which is tested in each experiment atseveral concentrations to generate a concentration-response curve fromwhich its IC₅₀ value is calculated.

EP₃ Receptor Agonism Assay (Inhibition of Forskolin-induced cAMPGeneration Assay)

Functional Assay #3AG

To test the ability of compounds to activate the EP₃ receptor, thedecrease in cAMP accumulation induced by forskolin following treatmentwith compounds is measured. Cells expressing the EP₃ receptor are platedin 24-well plates in normal growth medium and allowed to come toconfluence. When the cells have come to confluence, the medium isreplaced with 450 μl of serum-free medium containing 0.25 mM IBMX and 20μM indomethacin. Cells are incubated in this medium for one hour. Fiftymicroliters of this same buffer containing 3 μM forskolin and variousconcentrations of PGE₂ or compounds to be tested are subsequently addedto the cells. After incubation at 37° C. for 10 minutes, reactions areterminated by the addition of 500 μl of 10% TCA. cAMP measurements ofthe cell lysates are performed using Cayman Chemical's cAMP EIA Kitfollowing the instructions provided in the kit booklet.

Alternative EP₄ Receptor Agonism Assay

Functional Assay #4AGii

(Cerep, Catalog Reference 758-49a; Wilson, R. J., et al., Ibid.)

Evaluation of the agonist activity of compounds at the human EP₄receptor in transfected CHO cells, determined by measuring their effectson cAMP production using the HTRF detection method.

The cells are suspended in HBSS buffer (Invitrogen) complemented withHEPES 20 mM (pH 7.4) and 500 μM IBMX, then distributed in microplates ata density of 2·10⁴ cells/well and incubated for 10 minutes at roomtemperature in the absence (control) or presence of the test compound orthe reference agonist. For stimulated control measurements, separateassay wells contain 1 μM PGE₂. Following incubation, the cells are lysedand the fluorescence acceptor (D2-labeled cAMP) and fluorescence donor(anti-cAMP antibody labeled with europium cryptate) are added. After 60minutes at room temperature, the fluorescence transfer is measured atλex=337 nm and λem=620 and 665 nm using a microplate reader (Rubystar,BMG). The cAMP concentration is determined by dividing the signalmeasured at 665 nm by that measured at 620 nm (ratio). The results areexpressed as a percent of the control response to 1 μM PGE₂. Thestandard reference agonist is PGE₂, which is tested in each experimentat several concentrations to generate a concentration-response curvefrom which its EC₅₀ value is calculated.

EP₄ Receptor Antagonism Assay

Functional Assay #4ANT

(Cerep, Catalog reference 758-49b; Wilson, R. J., et al., Ibid.)

Evaluation of the antagonist activity of compounds at the human EP₄receptor in transfected CHO cells, determined by measuring their effectson agonist-induced cAMP production using the HTRF detection method.

The cells are suspended in HBSS buffer (Invitrogen) complemented withHEPES 20 mM (pH 7.4) and 500 μM IBMX, then distributed in microplates ata density of 2·10⁴ cells/well and preincubated for 5 minutes at roomtemperature in the absence (control) or presence of the test compound orthe reference antagonist.

Thereafter, the reference agonist PGE₂ is added at a final concentrationof 10 nM. For basal control measurements, separate assay wells do notcontain PGE₂. Following 10 minutes incubation at room temperature, thecells are lysed and the fluorescence acceptor (D2-labeled cAMP) andfluorescence donor (anti-cAMP antibody labeled with europium cryptate)are added. After 60 minutes at room temperature, the fluorescencetransfer is measured at λex=337 nm and λem=620 and 665 nm using amicroplate reader (Rubystar, BMG). The cAMP concentration is determinedby dividing the signal measured at 665 nm by that measured at 620 nm(ratio). The results are expressed as a percent inhibition of thecontrol response to 10 nM PGE₂. There is no standard referenceantagonist for this assay.

1. A compound of the general formula (I):

wherein dashed bonds may each independently represent a secondcarbon-carbon bond in order to give a carbon-carbon double bond witheither (E) or (Z) geometry or may be ignored in order to give acarbon-carbon single bond; wherein C⁹ and C¹¹ each is independentlyC═CH₂, C═O, CF₂, CHF (any stereoisomer), or C(H)OH (any stereoisomer)with the proviso that C⁹ does not equal C¹¹, and also with the provisothat when one of either C⁹ or C¹¹ is C═O, and the other is C(H)OH, atleast one of Z², Z³, Z⁴, and Z⁵ is fluorine, and also with the provisothat when one of either C⁹ or C¹¹ is CHF, the other is not C(H)OH;wherein R¹ is CO₂R³, CH₂OR³, CONR⁴R⁵, COCH₂OH, CONR⁴SO₂R⁵, P(O)(OR⁴)₂,or

wherein R³ is hydrogen or (C₁-C₆)-alkyl: wherein R⁴ and R⁵ each isindependently hydrogen or (C₁-C₆)-alkyl; wherein Z¹ are hydrogen orfluorine; wherein Z² and Z³ each is independently hydrogen or fluorine;wherein Z⁴ and Z⁵ each is independently hydrogen, fluorine, hydroxy, ormethyl, or together are an oxygen atom that form a carbonyl group withthe adjoining carbon atom of the ω chain; and wherein Z⁶ and Z⁷ each isindependently hydrogen, fluorine, hydroxy, or methyl, or together are anoxygen atom that form a carbonyl group with the adjoining carbon atom ofthe ω chain; or any stereoisomer of the compound of the general formula(I), or any geometric isomer of the compound of the general formula (I),or an equivalent of the compound of the general formula (I), or aprodrug of the compound of the general formula (I), or a hydrate of thecompound of the general formula (I), or a solvate of the compound of thegeneral formula (I), or a pharmaceutically acceptable salt of thecompound of the general formula (I).
 2. The compound of claim 1, whereinthe compound of the general formula (I) comprises the compound ofgeneral formula (II):


3. The compound of claim 1, wherein the compound of the general formula(I) comprises the compound of general formula (III):


4. The compound of claim 1, wherein the compound of the general formula(I) comprises the compound of general formula (IV):


5. The compound of claim 1, wherein the compound of the general formula(I) comprises the compound of general formula (V):


6. A pharmaceutical composition comprising a pharmaceutically effectiveamount of a compound of claim 1 in admixture with a pharmaceuticallyacceptable carrier.
 7. A method of expanding hematopoietic stem cellpopulations in a culture or patient in need thereof comprisingadministering to the culture or the patient a compound according toclaim
 1. 8. A method of treatment for a patient comprising administeringto the patient a compound according to claim
 1. 9. The method of claim8, wherein said compound further comprises a pharmaceutically acceptablecarrier.
 10. A method for treating or preventing EP receptor-mediatedconditions in a subject, comprising the step of administering to thesubject a compound according to claim 1.